Method for assaying sepsis in humans

ABSTRACT

The present invention relates to a reliable method of prediction of sepsis in humans after a trauma, wherein the level of pancreatic stone protein/regenerating protein (PSP/reg) is determined in serum, and a high level is indicative of the development of sepsis at early stages of the disease. Furthermore a method of determination of PSP/reg levels in serum is described.

CROSS REFERENCE TO RELATED APPLICATIONS

This is a continuation application claiming benefit of U.S. applicationSer. No. 12/676,650 filed Sep. 2, 2008, which is incorporated herein byreference in its entirety and which is U.S. national stage ofInternational application no. PCT/EP2008/007158, filed Sep. 2, 2008designating the United States and which claims priority to Europeanapplication no. 07017539.3, filed Sep. 7, 2007.

REFERENCE TO SEQUENCE LISTING

The Sequence Listing submitted herewith as a text file named“3023-122CON-Sequence-Listing,” created on Mar. 29, 2013, and having asize of 1594 bytes is hereby incorporated by reference pursuant to 37C.F.R.1.52(e)(5).

FIELD OF THE INVENTION

The present invention relates to a method of prediction and/or diagnosisof a systematic infection in human, in particular for prediction of thedevelopment of sepsis, based on the level of pancreatic stoneprotein/regenerating protein (PSP/reg) in body fluids, and to an assaykit.

BACKGROUND OF THE INVENTION

Systemic responses to severe trauma include a number of parametersaffecting innate immunity, inflammatory reactions and cellularactivities. Severe trauma patients may have a benign outcome with noinfection while others suffer from infections or sepsis. Sepsis isassociated with multiple organ failure and a high mortality. Among themost commonly used markers for diagnosis of sepsis are leukocyte counts,C-reactive protein and procalcitonin. The latter are two proteins highlyinduced after trauma, yet without any known function. In addition,cytokines such as IL-6, IL-8 and IL-18 are employed to monitor patients.However, none of the above mentioned markers serves as a predictiveindicator for infections including sepsis, hence treatment may lagbehind the onset of sepsis.

In animal pilot experiments, it has been shown that a pancreatic proteinis induced due to handling stress, even in the absence of pancreatictissue damage (R. Graf et al., J Surg Res 2002, 195:136-144). Thisprotein, pancreatic stone protein/regenerating protein (PSP/reg) belongsto a family of lectin binding proteins. Under conditions of acute orchronic pancreatitis, it is highly up-regulated and may appear in theserum. Since the regulation of this protein is not purely restricted todiet induced secretion like other zymogens, it may appear elevated inother conditions, e.g. during pancreatitis. Thus far, the protein hasbeen studied predominantly in the pancreas. It is also synthesized inPANETH cells of the small intestine and the fundic cells of the stomach.The function of PSP/reg is still highly debated, but it is generallyassumed that it is involved in promoting cell proliferation duringregenerative processes (Y. Kinoshita et al., J. Gastroenterol 2004,39:507-513).

Several efforts have been made to establish PSP/reg as a disease marker.So far, it has not been possible to establish a correlation of serumvalues with a specific disease entity since serum levels are raised invarious gastrointestinal diseases Y (Satomura et al., J Gastroenterol1995, 30:643-650).

SUMMARY OF THE INVENTION

The present invention relates to a method of prediction and/or diagnosisof a systemic infection in humans, in particular for prediction of thedevelopment of sepsis, wherein the level of pancreatic stoneprotein/regenerating protein (PSP/reg) is determined in body fluidsample, and a high level is indicative of the development of sepsis atearly stages of the disease. In addition, the present invention relatesto a method of determination of PSP/reg levels in body fluids, and a kitof parts for such a method.

BRIEF DESCRIPTION OF THE FIGURES

FIG. 1: Determination of C-reactive protein (CRP) values in sera ofpatients after admission to the hospital with a severe trauma.

Patients were retrospectively categorized: no infection (open boxes,n=14), patients with infection (lightly hatched boxes, n=22), patientswith sepsis (darkly hatched boxes, n=27). CRP values (log₁₀ ng/mL) ofthe three groups are plotted as box plots with the mean and the 95%confidence interval. d=days after trauma. Statistical analysis wasperformed using multivariate analysis; p=significance, *=p values forsepsis vs. no infection.

FIG. 2: Determination of IL-6 values in sera of patients after admissionto the hospital with a severe trauma.

Patient categorization and presentation of IL-6 values (log₁₀ pg/mL) ina box blot as for CRP values in FIG. 1.

FIG. 3: Determination of procalcitonin (PCT) values in sera of patientsafter admission to the hospital with a severe trauma.

Patient categorization and presentation of PCT values (log₁₀ ng/mL) in abox blot as for CRP values in FIG. 1.

FIG. 4: Determination of pancreatic stone protein/regenerating protein(PSP)

Temporal profile of PSP/reg after a trauma at day 0. All values werecombined for each time point. C (=control) indicates value for healthysubjects. d=days after trauma.

FIG. 5: Determination of PSP/reg values in sera of patients afteradmission to the hospital with a severe trauma.

FIG. 5A: Patient categorization and meaning of open, lightly hatched anddarkly hatched boxes as for CRP values in FIG. 1. PSP/reg values (ng/mL)of the three groups are plotted as mean +/−SEM. d=days after trauma.

FIG. 5B: Patient categorization and presentation of PSP/reg values(ng/mL) in a box blot as for CRP values in FIG. 1. p=significance, *=pvalues for sepsis vs. no infection.

DETAILED DESCRIPTION OF THE INVENTION

The present invention relates to a method of prediction and/or diagnosisof a systemic infection in humans, in particular for prediction of thedevelopment of sepsis, wherein the level of pancreatic stoneprotein/regenerating protein (PSP/reg) is determined in a body fluidsample, e.g. serum, and a high level is indicative of the development ofsepsis at early stages of the disease.

Other body fluids than serum useful for determination of PSP/reg levelsare e.g. whole blood, urine, sputum, cerebrospinal fluid, tear fluid,sweat, milk, or extracts from solid tissue or from fecal matter.

The PSP/reg level indicative for development of posttraumatic sepsis isdependent on the body fluid chosen for the determination. For bloodserum this level is 60 to 80 ng/ml on days 3, 4 or 5. Hence, morespecifically, the invention relates to a method of prediction and/ordiagnosis of the development of sepsis, wherein the level of pancreaticstone protein/regenerating protein (PSP/reg) is determined in serum, anda level of 60 ng/ml or more, in particular a level of 80 ng/ml or more,on days 3, 4 or 5 is indicative of the development of sepsis.

Any known method may be used for the determination of the level ofPSP/reg in body fluids. Methods considered are e.g. ELISA, RIA, EIA,mass spectrometry, or microarray analysis. Such methods when used fordiagnosis of systemic infection, e.g. sepsis, are a further object ofthe invention.

A preferred method for the determination of PSP/reg in human bodyfluids, e.g. serum, is an ELISA. In one embodiment of the invention, thePSP/reg ELISA consists of a sandwich array: Conventional microtiterplates are coated with one type of antibody (“first” antibody”), e.g. aguinea pig polyclonal antibody, directed against PSP/reg. The plates arethen blocked and the sample or standard is loaded. After the incubation,a different type of antibody (“second” antibody) against PSP/reg isapplied, e.g. a polyclonal rabbit antibody. A third antibody detectingthe particular type of the “second” antibody, e.g. a anti-rabbitantibody, conjugated with a suitable label, e.g. an enzyme forchromogenic detection, is then added. Finally the plate is developedwith a substrate for the label in order to detect and quantify thelabel, being a measure for the presence and amount of PSP/reg. If thelabel is an enzyme for chromogenic detection, the substrate is acolour-generating substrate of the conjugated enzyme. The colourreaction is then detected in a microplate reader and compared tostandards.

Suitable pairs of antibodies (“first” and “second” antibody) are anycombination of guinea pig, rat, mouse, rabbit, goat, chicken, donkey orhorse. Preferred are polyclonal antibodies, but it is also possible touse monoclonal antibodies or antibody fragments. Suitable labels arechromogenic labels, i.e. enzymes which can be used to convert asubstrate to a detectable coloured or fluorescent compound,spectroscopic labels, e.g. fluorescent labels or labels presenting avisible colour, affinity labels which may be developed by a furthercompound specific for the label and allowing easy detection andquantification, or any other label used in standard ELISA.

Other preferred methods of PSP/reg detection are radioimmunoassay orcompetitive immunoassay using a single antibody and chemiluminescencedetection on automated commercial analytical robots. Microparticleenhanced fluorescence, fluorescence polarized methodologies, or massspectrometry may also be used. Detection devices, e.g. microarrays, areuseful components as readout systems for PSP/reg.

The invention further relates to a kit of parts for the determination ofPSP/reg for diagnosis/prediction of systemic infection, for examplecomprising apparatus, reagents and standard solutions of PSP/reg.Apparatus considered are e.g. microtiter plates for ELISA, pre-coatedELISA plates, and plate covers. Reagents are those reagents particularlydeveloped and designed for the detection of PSP/reg. Standard solutionsof PSP/reg preferably contain PSP/reg synthesized according to thedirections hereinbelow. The kit of parts may contain further hardware,such as pipettes, solutions such as buffers, blocking solutions and thelike, filters, colour tables and directions for use.

PSP/reg is a protein expressed in the pancreas and the intestine. It canbe cloned from pancreatic mRNA and subcloned into a yeast expressionvector. The protein can then be expressed under the control of ADH. Asuitable expression medium may comprise methanol to induce and maintainthe secretion of PSP/reg. PSP/reg is preferably purified usingSP-Sepharose-cellulose by a pH and salt gradient. Such purified PSP/regis used to prepare standard solutions for comparison with PSP/reg levelsin body fluids. Polyclonal antibodies against the protein may beobtained from mice, rats, rabbits, goats, chicken, donkey, horses andguinea pigs or other suitable animals using standard methods.

The reason for the increase of PSP/reg in blood serum during earlydevelopment stages of sepsis is not entirely clear. In rat pilotexperiments an increase in PSP/reg synthesis in the absence ofpancreatic damage was observed, and there was evidence that significanttraumatic damage to other organs leads to an increase in blood levels ofPSP/reg. For further studies a set of human patients with severe traumabut apparent absence of pancreatic damage was chosen (see Examples). Theappearance of PSP/reg in blood serum would imply an altered pathway,diverting the protein from pancreatic juice into the blood. It has alsobeen shown that members of the lectin binding family (e.g.pancreatitis-associated protein) are inducible by cytokines. There is astrong and concerted action of cytokines after trauma. The complexity ofthe cytokine response, with many different cytokines being released, isnot understood. Thus it is likely that PSP/reg reacts to cytokines thatare raised under condition of systemic stress or trauma. In contrast,other pancreatic enzymes, e.g. amylase and lipase, appear not to beregulated by cytokines, their appearance in the blood being a result ofdiversion only. The PSP/reg level in blood serum is now proven to be areliable indicator of sepsis. The increase of PSP/reg in blood mightimply a specific stress response.

It is shown that unlike other indicators of inflammation, the level ofPSP/reg is highly increased in patients during or before clinical signsof sepsis are apparent. The detection and quantification of serumPSP/reg is accomplished e.g. by a sandwich ELISA with a limit ofdetection of less than 100 pg/ml. Normal serum values are between 5 and15 ng/ml. Patients with a severe trauma develop sepsis between day 7 and10 after the accident causing the trauma. The serum values correlatewith the severity of sepsis. They may reach over 200 ng/ml. Beforeclinical signs of sepsis are available, PSP/reg values start to increaseat day 3 to day 5 and reach values above 60-80 ng/ml. These values allowto predict whether a patient will develop sepsis and hence the need forintensive treatment including costly antibiotic treatment and a stay inthe intensive care unit. Compared to commercially available diagnosticassays, the PSP/reg ELISA is significantly better in monitoring putativeseptic patients.

EXAMPLES

Isolation and Subcloning of PSP/reg

In order to obtain cDNA for the production of PSP/reg specificantibodies, such cDNA is prepared by reverse transcription of pancreaticmRNA using state of the art laboratory methods. A PCR reaction usingprimers specific for the sequence coding for PSP/reg and selectivelyamplifying PSP/reg cDNA is performed. The PCR reaction is then repeatedwith the elongation primer to add a sequence specific for insertion intothe Pichia pastoris transfection vector. The primer is designed to fusethe coding region of the signal peptide of the alpha-mating factor witha KEX2 site and the coding region of the mature human PSP/reg.Subcloning into the Pichia pastoris vector is a two-step procedure.First the PCR product is ligated into the pCR2.1 vector (Invitrogen,TAcloning) and the sequence verified. Then the PCR product is cleaved byXhol/Notl restriction digestion and ligated into transfer vector pPIC9(Invitrogen). The Pichia pastoris strain KM71 (Invitrogen) istransformed and the most productive clone is selected for expansion andproduction of recombinant protein.

Primers used for PCR Amplification and Subcloning

Human PSP/reg/reg1 alpha Forward primers (SEQ ID NO: 1) 5′GAAAAGACAAGAGGCCCAGACAGAGTT 3′ (SEQ ID NO: 2) 5′GTATCTCTCGAGAAAAGACAAGAGGCCCAGA 3′ (elongation) Reverse (SEQ ID NO: 3)5′ CTAGTTTTTGAACTTGCATAC 3′ Human PSP/reg/reg1 beta Forward primers(SEQ ID NO: 4) 5′ GAAAAGACAGGAGTCCCAGACAGAGCTG 3′ (SEQ ID NO: 5) 5′GTATCTCTCGAGAAAAGACAGGAGTCCCAGAC 3′ (elongation) Reverse primer(SEQ ID NO: 6) 5′ ATCTGCAGTCTAGAATTCTGCAGGACCAGTTCTAGAC 3′

Large Scale Expression of Protein

Using a single colony, 25 ml of BMG (buffered minimal glycerol, 100 mMpotassium phosphate pH 6.0, 1.34% yeast nitrogen base, 4×10⁻⁵% biotin,1% glycerol) is inoculated in a 250 ml baffled flask and grown at 29° C.in a shaking incubator (300 rpm) overnight. 10 ml of this culture isused to inoculate 1 liter of BMG in a 3 liter baffled flask and grown at29° C. (300 rpm) overnight. The cells are harvested by centrifugation at1500-3000×g for 5 minutes at room temperature. Expression is induced byresuspending the cells in ⅕ volume (200 ml) of BMM (buffered minimummethanol, BMG in which glycerol is replaced by 0.5% methanol) in thesame baffled flask. 100% methanol is added to achieve a concentration of0.5% (1 ml) every 24 hrs until optimal time of induction is reached. Thecells are harvested by centrifugation at 1500-3000×g at roomtemperature. The medium supernatant is collected and frozen untilpurification of the peptide.

The polypeptide is purified from media supernatants. Media supernatantsare diluted 1:3 with distilled water. The pH is adjusted to pH 3.5 withHCl. The medium supernatant is then applied to a SP-Sepharose column andeluted by a salt and pH gradient (10 mM LiCI, 50 mM MES, pH 5.3 startingbuffer, 2 M LiCl, 50 mM MES, pH 6.3 end buffer). Fractions are collectedand analyzed by SDS-gel electrophoresis. The fractions with the highestand purest protein contents are combined and dialyzed against 10 mMHEPES pH 7.5. The sequence of the polypeptide is verified by N-terminalsequencing and the concentration is assessed by amino acid analysis.

PSP/reg ELISA

In order to determine total PSP/reg, a sandwich ELISA may be used on thebasis of a guinea pig antiserum raised against recombinant human PSP/regand a rabbit antiserum against the same protein. To improve thespecificity and sensitivity of the rabbit antibody, IgGs are purified byabsorption on a column of protein A beads (HiTrap®, Pharmacia): AHiTrap® column is equilibrated with 200 mM NaH₂PO₄/Na₂HPO₄ at pH 7. Therabbit antiserum is pH-adjusted with the same buffer solution (finalconcentration 20 mM) and then loaded onto the column, which isafterwards washed with 100 mM and 10 mM Tris/HCl pH 8 consecutively. TheIgG fraction is eluted with 0.1 M citric acid pH 3. The eluted fractionsare immediately neutralized with 1 M Tris/HCl pH 8.9.

96-well microtiter plates (Costar EIA plates, flat bottom, high binding)are coated over night at 4° C. with guinea pig anti-rat PSP/reg IgGfraction, diluted 1:500 in TBS (100 μl/well). After a washing step, theplate is blocked with 150 μl 1% BSA/TBS for one hour, which isafterwards replaced by 100 μl of different standard concentrations ofrecombinant human PSP/reg (0, 0.1, 0.5, 1.0, 1.5, 2.5, 3.5, or 5.0ng/ml) or 100 μl samples of diluted sample. Samples and standards areloaded in duplicates and incubated for 1 hr at room temperature. Afterrepeated washing, the plate is incubated for 1 hr with 100 μl rabbitanti-rat PSP/reg IgG, diluted 1:500. Another washing step follows beforea 30 min incubation with 100 μl of a commercially available mousemonoclonal anti-rabbit IgG antibody is started (mouse anti-rabbitalkaline phosphatase conjugated, IgG fraction, diluted 1:1000; purchasedfrom Sigma). The plate is then washed again, and a soluble phosphatasesubstrate, p-nitrophenyl phosphate disodium (Sigma 104® tablets), addedin alkaline phosphatase buffer (100 mM Tris/HCl pH 9.5, 100 mM NaCl, 0.8mM MgCl₂). After an incubation period of about 20 min optical density(OD) at 405 nm is measured in an MRX microplate reader (DynatechLaboratories).

All dilutions (except coating antibodies) are prepared in 1% BSA/TBS.All incubations at room temperature are carried out on a rotationalELISA plate shaker (Titramax 100, Heidolph, Bioblock Scientific). Allwashing steps are performed with TBS/Tween 20 (0.05%, v/v), using anautomatic microtiter plate washer (MRW, Dynatech Laboratories). Recoveryrates of recombinant PSP/reg into diluted serum from a healthy volunteeris as follows: 71% at 1:10, 118% at 1:20 and 95% at 1:40 dilution.Intraplate and interplate variance is less than 5% and 10%, respectivelyfor concentrations within the range of the standard (between 0.1 and 3.5ng/ml).

The test is established with recombinant human PSP/reg1 alpha.Recombinant PSP/reg1 beta, the second isoform, was made using the sametechnique. PSP/reg 1 beta is recognized equally well by the ELISA.Therefore, the ELISA is specific for the known PSP/reg family ofproteins.

Test Patients for Proof of Principle The study population included 63injured patients who were admitted to the Division of Trauma Surgery(level I trauma center), University Hospital Zurich, in a time periodfrom January 2002 to September 2003. Inclusion criteria were an injuryseverity score (ISS)>16 points, patient age>16 years, less than fourhours between accident and hospital admission, and surveillance on theintensive care unit (ICU) with a survival of more than five days.Patients with a pancreatic injury were excluded. All patients weretreated according to the advanced trauma life support (ATLS) guidelinesand a standard trauma protocol. In brief, after control of airway,ventilation, and monitoring of cardiovascular functions, life-savingprocedures including decompression of body cavities, control ofhemorrhage and contamination were carried out. This was followed byradical wound debridement, decompression of compartments, and primarystabilization of major fractures mostly through external fixation (“dayone surgery”). Thereafter, patients were transferred to the ICU forrestoration of organ functions. Of note, all patients received enteralnutrition within 24 hours after trauma to maintain normal intestinalflora and bowel mucosa. Antibiotics were used, if a septic focus wasverified by a positive bacterial culture. In addition, for openfractures standard antibiotics are applied for five days and a singleshot of a cephalosporin was given as prophylaxis for osteosynthesis offractures.

Table 1 summarizes demographic data and injury scores at the day ofadmission. The severity of injury and gender distribution were verysimilar.

TABLE 1 Demographic data of enrolled patients Parameter No infectionInfection Sepsis Number 14 22 27 Age (yr) 38.6 ± 16.9 36.6 ± 15.5 37.3 ±16.0 Males 11 (78.6%) 15 (68.2%) 22 (81.5%) ISS^(a) (points) 34.6 ± 9.3 32.2 ± 13.9 38.7 ± 15.6 GCS^(b) (points) 8.9 ± 5.2 9.2 ± 4.9 8.8 ± 5.1APACHE II^(c) (points) 15.0 ± 6.2  13.6 ± 6.6  17.0 ± 6.8  ICU^(d)(days) 9.1 ± 5.9 16.3 ± 8.2  26.6 ± 9.9  Mean ± SD. Values inparentheses are percentages. ^(a)ISS, injury severity score ^(b)GCS,Glasgow coma scale ^(c)APACHE II, acute physiology and chronic healthevaluation II ^(d)ICU, intensive care unit.

Blood Status of Trauma Patients

Patients are retrospectively assigned to three groups depending on theirscore: a) no infection, b) infection and c) sepsis (Table 2). Todemonstrate the course of several parameters used to determine theextent of inflammation and injury, blood leukocyte counts and C-reactiveprotein were determined. All patients exhibited a strong reduction ofblood leukocytes at day one of hospitalization, irrespective of theseverity group.

Leukocytes gradually increase to normal with the exception of septicpatients which reach a significantly higher leukocyte count of 18×10⁶/Lat day 10. Concurrent determination of C-reactive protein (CRP) indicatea gradual increase from low levels at admission to about 150 ng/ml atday three in all groups (FIG. 1). Although the non-infected group hasconsistently lower levels than the other groups, there is no obviouspattern that distinguishes the three patient groups. Thus between day 5and day 7 and between day 14 and 21, septic patients exhibit a higherCRP than non-septic patients, the difference being less than a factor oftwo.

TABLE 2 Injury pattern and posttraumatic course of enrolled patients Noinfection Infection Sepsis Parameter (n = 14) (n = 22) (n = 27) Head(AIS ^(a), points) 85.7% (3.4) 86.4% (3.7) 70.4% (4.1) Thorax (AIS ^(a);points) 78.6% (3.0) 36.4% (3.3) 63.0% (3.5) Abdomen (AIS ^(a); points)50.0% (3.7) 36.4% (3.8) 44.4% (4.1) Extremities (AIS ^(a); points) 57.1%(3.0) 68.2% (2.3) 63.0% (2.5) Pelvis (AIS ^(a); points) 21.4% (3.0)22.7% (2.6) 18.5% (2.8) Spine (AIS ^(a); points) 42.9% (2.8) 36.4% (2.5)25.9% (2.9) No SIRS ^(b) 2 (14.3%) — — SIRS 2 ^(b) 5 (35.7%)  2 (9.1%) —SIRS 3/4 ^(b) 7 (50.0%) 20 (90.9%) — Sepsis — — 27 (100%) Mortality 2(14.3%)  2 (9.1%)  5 (18.5%) ^(a) AIS, abbreviated injury scale ^(b)SIRS, systemic inflammatory response syndrome

Determination of Standard Indicators of Inflammation in Trauma Patients

To determine whether commonly used indicators of inflammation, e.g. IL-6(FIG. 2) and procalcitonin (PCT, FIG. 3), could distinguish between thethree severity groups, blood levels of these proteins were measuredduring the whole course of the hospital stay. IL-6 immediately increasedprostraumatically reaching the highest levels at day 1. During the firsttwo days the three severity groups are different, with the septic group(1200 pg/ml) and the infected group (600 pg/ml) higher than thenon-infected group. The statistics do not indicate significance due tothe high variability of the data while at day 5-10 there was adifference. Although there is a slight increase during the time ofsepsis (350 pg/ml) these levels are low in comparison to the first dayof hospitalization.

PCT was clearly increased 20-fold in the septic patient group prior toand during sepsis, while the other groups remain at levels around 0.5-2ng/ml (FIG. 3). The maximal increase in PCT was 25 fold versus healthysubjects. The statistics do not indicate significant differences due tothe high variation in the sample.

PSP/reg is Upregulated Posttraumatically

PSP/reg is synthesized predominantly in the pancreas and in theintestine. In response to a local tissue injury it is highlyupregulated. In the absence of pancreatic tissue injury, PSP/reg is notexpected to be up-regulated. However, polytrauma causes a release ofcytokines that may affect the expression and secretion of PSP/reg.Therefore, it was tested whether these proteins are increased inpatients with severe trauma that had no pancreatic injury. The combineddata of all patients after polytrauma indicate an increase at day onethat becomes significant at day three, compared to day zero as well ascompared to healthy subjects (FIG. 4).

Thus, pancreatic stone protein is slightly increased after a polytrauma.The data were then analyzed using stratification assigned to patientswithout infection, patients with infection and patients with sepsis.PSP/reg values in patients without an infection are slightly increasedwith a peak at day 7-10 (FIGS. 5A, 5B). In patients with polytrauma thatexhibit infection, there is a further increase. Finally, polytraumaticpatients exhibiting sepsis show a large increase in serum PSP/reg.

The increase starts several days before clinical criteria of sepsis arefulfilled. PSP/reg highly correlates with sepsis. At day 3, whenpatients are not septic yet the average level of PSP in blood increasessignificantly to over 100 ng/ml and reaches about 20-fold during thetime of sepsis (FIGS. 5A, 5B, day 5-10).

The early increase of PSP/reg in patients with sepsis can therefore beused as a serum marker to predict sepsis. Therefore the specificity, thepositive and the negative predictive values are summarized for threepotential cut-off values e.g. 30, 60 and 80 ng/ml at day three and five.The specificity is around 80 percent for cut-off values of 60 and 80ng/ml. The positive and negative predictive values above are alsobetween 60 and 80% indicating that patients can be identified by thismethod early on.

TABLE 3 Sensitivity, specificity, and positive and negative predictivevalues for three cut off points of PSP serum levels for patients withsepsis compared with patients without infectious complications or localinfections. The two groups non-infection were compared with the septicgroup. The infectious group is not included. 30 ng/mL 60 ng/mL 80 ng/mLDay 3 Sensitiviy (%) 70.4 55.5 40.7 Specificity (%) 72.2 83.3 83.3Positive predictive value (%) 65.5 71.4 64.7 Negative predictive value(%) 76.5 71.4 65.2 Day 5 Sensitiviy (%) 74.1 63.0 51.9 Specificity (%)66.7 75.0 77.7 Positive predictive value (%) 62.5 65.4 63.6 Negativepredictive value (%) 77.4 73.0 68.3

The analysis is based on PSP serum values obtained on day 3 or day 5after trauma.

1.-7. (canceled)
 8. A kit of parts for the determination of PSP/reg forprediction and/or diagnosis of a systemic infection in humans comprisingapparatus, reagents and standard solutions of PSP/reg, wherein the levelof pancreatic stone protein/regenerating protein (PSP/reg) is determinedin a body fluid sample, and a level above 60 ng/ml is indicative of thedevelopment of a sepsis.
 9. A method of diagnosis of sepsis after traumain a human comprising: (a) quantifying a level of pancreatic stoneprotein/regenerating protein (PSP/reg) in a serum sample of said humanon days 3, 4 or 5 after said trauma, (b) determining whether the levelof PSP/reg quantified in said sample is above 60 ng/ml, and (c)diagnosing sepsis when said PSP/reg quantified in said serum sample isabove 60 ng/ml.
 10. The method of claim 9, wherein, upon diagnosingsepsis in said human, providing treatment for said human against sepsis.11. The method of claim 10, wherein said treatment comprises antibiotictreatment.
 12. The method of claim 9, wherein said human is monitoredfor sepsis.
 13. The method of claim 9, wherein the level of PSP/reg isabove 80 ng/ml.
 14. The method of claim 9, wherein the level of PSP/regis determined by ELISA, RIA, EIA, mass spectrometry, or microarrayanalysis.
 15. The method of claim 9, wherein the level of PSP/reg isdetermined via a sandwich ELISA, comprising: providing microtiter platesare coated with a first antibody directed against PSP/reg, blocking saidplates, loading the sample or a standard, applying a second antibodyagainst PSP/reg, adding a third antibody that detect the secondantibody, wherein said third antibody is conjugated with a label is thenadded, and adding a substrate for the label to detect and quantify thelabel.
 16. The method of claim 15, wherein the label is an enzyme forchromogenic detection and the substrate is substrate is acolor-generating substrate of the conjugated enzyme.
 17. The method ofclaim 15, wherein the first and second antibody are polyclonalantibodies.